The average sperm nucleus is only 8 micrometers long, but contains 1 meter of DNA. We have previously described how the sperm cell is able to package this DNA into the small volume of the sperm head. The only DNA structure that exists in both somatic cells and sperm nuclei is the organization of DNA loop domains that are attached at their bases to the nuclear skeleton, the nuclear matrix. We compared the structure of one gene in sperm nuclei to that of spermatogenic and adult cells.

Individual loop domains of the 5S rRNA gene cluster were visualized by fluorescent in situ hybridization (FISH). Nuclei were isolated from hamster spermatozoa, spermatogonia, pachytene spermatocytes, round spermatids, and adult liver cells. They were then extracted to remove the protamines or histones, fixed, hybridized to a biotinylated 5S rDNA probe, then viewed by epifluorescence microscopy. The 5S rDNA in liver nuclei was organized into a single large loop domain (Fig. 1),

Environmental Scanning Electron Microscopy has opened up the potential to study biological events in their native occurence. While still in the early stages of exploration, the ability to view samples in their native state with the resolution provided by scanning electron microscopy is highly desirable and will provide insights in dynamic processes which previously had only been possible by interpretation with indirect methods or by static images obtained with transmission electron microscopy.

In preliminary experiments to explore the potential of studying native events with the ESEM we have investigated sea urchin embryos after deciliation with chloral hydrate. The Philips XL-30 ESEM instrument was used to obtain images of lightly fixed (0.1% glutaraldehyde in sea water), lightly coated embryos that had been treated as follows. Chloral hydrate was added at a concentration of 0.1% to blastulae of Lytechinus pictus at 24 hours after fertilization. Observation with light microscopy indicates that cilia become gradually shed into the sea water within 10 to 20 hours after chloral hydrate application.

Sea urchins have been used for over a century as a remarkable animal model system in which to study cell, molecular, and developmental biology. The studies presented here have used sea urchin eggs and embryos for pioneering experiments to explore the effects of microgravity on the cytoskeleton during a space flight on the space shuttle Endeavor. The culture conditions followed those described previously utilizing the Aquatic Research Facility (ARF) to fertilize and culture eggs and embryos up to the pluteus stage under controlled temperature (12°C) and fixation conditions. To achieve a final fixation with 0.5% glutaraldehyde and 4μM taxol, concentrated fixation fluid was injected at preselected time points to preserve microtubules, centrioles, centrosomes, microfilaments, mitochondria, and cell membranes.

The analysis of the results revealed that the centriole-centrosome complex during cell division and cilia formation showed alterations in samples that had been exposed to microgravity while control cells cultured in a centrifuge at lg in space and those cultured on ground appeared normal.

An integrated approach including TEM, SEM and LM was utilized to investigate adhesives produced by the marine fouling diatom Achnanthes longipes. Extracellular adhesive secretion during sequential attachment in living diatom cells was examined using video microscopy. A suite of cryotechniques including high pressure freezing (HPF), freeze-substitution (FS) and high resolution cryo-field-emission SEM (cryo-FESEM) were required for preservation and structural investigation of the hydrophilic polymers synthesized by A. longipes that promote attachment to ship hulls, oil rigs and other submerged structures.

Living cells and associated extracellular biocomposites were rapidly cryo-immoblized and observed using a Hitachi S-900 FESEM equipped with a cryo-stage. This technique allowed resolution of details of polymer organization within the pad, shaft and collar portions of the major adhesive structure termed a stalk (Fig. 1,2). This stalk has been demonstrated to consist mainly of polysaccharide cross-linked by O-linked polypeptides. In addition to permanently adhering the diatom to the substratum, the stalk elevates A. longipes, possibly enhancing the ability to successfully compete with other biofilm dwellers.

Freeze substitution techniques can provide exceptional fixation of many types of small biological materials. Ultrastructural studies on the Foraminiferida (marine rhizopods) traditionally have used standard chemical fixation protocols. The relatively large size (-25 μm - several cm) and the presence of a mineralized shell in most taxa precludes the application of many cryo-techniques. High pressure freezing, however, provides a method for freezing organisms as large as many of the smaller foraminifera often without extensive ice damage. Gamonts (sexual generation) from three representatives of the suborder Astrorhizina ﹛Myxotheca sp., Cribrothalammina alba, and Hyperammina sp.) were selected because they are among the most primitive foraminifera and have only very finely agglutinated shells with non-mineralized, organic cements. Myxotheca sp. and C. alba were originally collected from salt marsh environments on Sapelo Island, Georgia, and Hyperammina sp. was isolated from fine-grained carbonate sediments of the Florida Keys.

Crustacean hemocytes have been characterized on the basis of cell size and nature of cytoplasmic granules. Based on light microscopic morphological analysis and cytochemistry, investigators variously named the hemocyte types (agranular, small-granule, large granule, undifferentiated, hyaline cells, non-explosive, explosive granulocytes, etc.). In his study of the isopod Armadillidium vulgare Faso adopted the terminology of Benjamin and James and referred to the hemocytes as hyaline cells, semi-granulocytes and granulocytes.

In the present investigation we have studied the hemocytes of two isopods, Oniscus asellus and Armadillidium nasatum, using transmission electron microscopy. Hemolymph was collected by penetrating the posterior dorsal exoskeleton of 20 animals of each genus with a microcapillary pipette and drawing 3-5μL per isopod. The samples were processed following a standard technique. Thin sections were collected on 300 mesh copper grids, counterstained with 2% aqueous uranyl acetate and lead citrate, and viewed with a JEOL 1010 electron microscope.

Most plant pathogenic fungi that are obligate parasites produce haustoria which are thought to be involved in nutrient absorption. A haustorium is a specialized hyphal branch that penetrates the host cell wall and invaginates the host cell plasma membrane. The host plasma membrane ensheathing the haustorium is termed the extrahaustorial membrane. This presentation provides examples of different types of haustoria produced by plant pathogenic fungi. Species considered here are 1) Cronartium quercuum f. sp.fusiforme, the cause of fusiform gall rust of pine, 2) Puccinia arachidis, the cause of peanut rust1, 3) Uncinuliella australiana, the cause of powdery mildew of crape myrtle, 4) Exobasidium camelliae, a pathogen of Camellia sasanqua2, and 5) Cercosporidium personatum, the cause of late leaf spot of peanut.

Rust fungi typically require two different host species to complete their life cycles. The dikaryotic phase of the rust life cycle consists of intercellular hyphae that give rise to specialized haustoria known as D-haustoria which are remarkably similar from one species to the next.

The filamentous fungus Aspergillus nidulans partitions its cells by laying down septa at regularly spaced intervals in response to nuclear division. Physiological and genetic studies of the temperature-sensitive sep mutants have been especially useful in dissecting the regulation of septation. Electron microscopic studies of the sep mutants should be equally useful in dissecting the structural intermediates of septation. In preparation for ultrastructural studies of the sep mutants, we have examined septa in wild-type A. nidulans fixed by freeze substitution.

Dialysis membranes were placed on rich medium plates and inoculated with A. nidulans spore suspensions. After 12 hours at 30°C, the dialysis membranes with adhering fungal hyphae were cut into square pieces measuring approximately 5mm on each side. The pieces were plunged into liquid propane and processed according to the procedures of Hoch. Serial sections were cut using a diamond knife and post stained with uranyl acetate and lead citrate.

Ivermectin is a drug widely utilized for its anthelminthic activity over a diverse range of animal parasites. It has proved to be particularly useful in the prophylaxis of infection by the heartworm (Dirofilaria immitis) in dogs and cats. Although its application in this respect has been as a filaricide in preventing early growth and maturation of naturally acquired larvae, it is known to have activity against young adults as well. Previous studies have shown that in vitro exposure to ivermectin induces somatic muscle paralysis in the nematode Haemonchus contortus, resulting in pharyngeal dysfunction and disruption of normal ingestion. Experiments were performed to determine the effect of in vivo exposure of adult canine heartworms to this drug.

Adult heartworms were harvested from groups of dogs treated monthly with ivermectin beginning four to five months after inoculation of infective larvae and from untreated control dogs. Live worms from both experimental and control dogs were fixed, embedded, and sectioned for conventional transmission electron microscopy.

Perkinsus marinus is a protistan parasite that infects the Eastern Oyster, Crassostrea virginica, and is one factor responsible for the reduced productivity of oyster fisheries. This parasite (or related species) has a wide distribution and has also been found to infect the oysters in the Tampa Bay region. A significant aspect of this organism has been its enigmatic taxonomic position. Originally classified in two separate fungal genera, it currently is considered an Apicomplexan (Kingdom Protista) based on transmission electron microscopy studies of the zoospore revealing an apical complex. However, rRNA studies have shown that P. marinus may be more closely related to the dinoflagellates. The focus of my study has been on surface features of certain developmental stages that may aid in a definitive taxonomic placement of this organism.

Transmission electron microscopy observations of the trophozoite and hypnospore stages show variation in both the size of the organism and the presence of a cell wall.

Adult Rhabdochona nematodes, commonly parasitizing fish, were present in the digestive tracts of cutthroat trout in Little Cottonwood Creek, Utah. Cutthroat trout, Oncorhyncus clarki, are known to serve as both intermediate and definitive hosts for parasitic nematodes. The larval stage parasitizes almost any tissue of its host, but the adult is always found in the digestive tract. Due to the lack of key morphological features, scanning electron microscopy (SEM) was used to identify specific structures leading to the nematode's taxonomic identification.

Cutthroat trout were obtained using a rod and reel and were dissected the same day. Nematodes were present in all 12 cutthroat trout residing in all parts of the digestive tract. The nematodes, Rhabdochona sp., were prepared for SEM using the following procedures. First, the parasites were fixed in 2% buffered glutaraldehyde, washed in sodium cacodylate buffer, and post fixed in a 1% solution of osmium tetroxide. The samples were then washed in the same buffer system and dehydrated through a graded alcohol series. Critical-point-drying removed the remaining fluids. Finally, the nematodes were placed on specimen stubs, sputter coated with gold, and each specimen examined with a JOEL-840 high resolution scanning electron microscope with micrographs taken at varying magnifications.

Absorption enhancing microtriches of Bothriocephalus sp. were studied in relation to location, size, density, structure, and possible function using SEM.

Carp, C. carpio, were collected by gill nets set in Lake Powell, Utah. Tapeworms were collected from the carp intestine and relaxed in a cold water bath for 12 hours, placed in 1% cacodylate buffered glutaraldehyde, washed in a cacodylate buffer, soaked overnight in 1% OsO4, washed in buffer, dehydrated in an ETOH series, critical point dried, attached to stubs, and sputter coated for four minutes with gold. Bothriocephalus was viewed at varying magnifications using the JEOL 6100 scanning electron microscope. Density counts and measurements were conducted using digitized images and the program Semafore. Images were collected from the scolex and 8 equal body sections. Microtriches were measured from the tip to the base.

The ciliated protozoan Hyalophysa is a symbiont of a variety of crustaceans, including grass shrimp, hermit crabs, and blue crabs. At the molting of the host, the attached protozoans excyst and swim into the exoskeleton to feed by pinocytosis on exuvial fluid. The feeding trophonts expand to many times their original size, transforming into cells that are little more than large food vacuoles with a rim of cytoplasm stretched around the perimeter. After feeding for the only time during their life cycle, they settle on the substrate to encyst and divide into daughter tomites, and in doing so they divide the food vacuole. The tomites then leave the cyst and attach to a crustacean for their next opportunity to feed at molting.

We have followed endocytosis in Hyalophysa using two dyes, nigrosin and congo red, in order to understand the digestive process.

The standard organic coatings applied to Navy aircraft consist of an epoxy primer and a polyurethane topcoat. The old coatings are routinely stripped in order for new layers to be applied. The stripping process involves removing the coating by sandblasting the aircraft with small plastic pellets. Although unpublished, we have demonstrated an ability of Pseudomonas sp. VM1 to biodegrade the waste products associated with the sandblasting process. A primary objective of this investigation was to determine the growth kinetics associated with the utilization of paint waste products as a sole carbon source for Pseudomonas. Another objective was to ascertain the nature of morphologic changes that occur among the particles of paint during biodegradation. In addition, the study was designed to determine whether phenylmethylsulfonyl fluoride (PMSF), a recognized inhibitor of proteolytic enzymes, is capable of modifying the biodegradative process of paint waste by Pseudomonas.

The small size of many parasitic organisms requires the use of election microscopy for adequate elucidation of their structure. While both transmission and scanning electron microscopy can provide complementary results which allow considerable degree of structural correlation, each technique has its inherent limitations. Since previous studies have demonstrated that soft X-ray microscopy could be used to study parasitic protozoa and provide new information, the objective of this study was to determine whether soft X-ray microscopy could also be used to elucidate the morphology of small metazoa to complement the data obtained by other microscopy techniques.

Newborn larvae, approximately 7 μm × 110 μm in size, of parasitic nematode Trichinella spiralis were used as a model system. Some of the larvae, deposited by adult females maintained in vitro, were isolated and processed for examination by transmission and scanning electron microscopy as described in our previous studies, others were fixed in 4% glutaraldehyde (Millonig's buffer) and examined in the X-ray microscope XM-1, and in the BioRad MRC 1024 confocal laser (krypton/argon) microscope of the Advanced Light Source, Berkeley National Laboratory.

The near epidemic occurrence of fibropapilloma in green sea turtle (Chelonia my das) (Figure 1) significantly threatens the survival of this species which is protected under the U.S. Endangered Species Act. Although collective evidence suggests a viral etiology, the causative virus of green sea turtle fibropapilloma has not been isolated. To facilitate the isolation and characterization of the causative virus(es), we established 13 cell lines from multiple organs/tissues (tumor, kidney, lung, heart, gall bladder, testis, and skin) of green sea turtles with fibropapilloma. Serial subcultivation of cell lines derived from lungs, testis, eye soft tissues and tumors resulted in the formation of tumor-like aggregates, which attained sizes of 1-2 mm in diameter within two weeks (Figure 2). Media from such cultures, when inoculated onto cells derived from healthy turtle embryos, produced similar tumor-like aggregates, suggesting the presence of a transmissible agent.

Upon thrombin-catalyzed proteolytic conversion of fibrinogen to fibrin, fibrinopeptide A release exposes an N-terminal polymerization site (‘EA’) in the central E domain that interacts with a constitutive complementary site in each outer D domain (‘Da’) to drive the molecular assembly process by end-to-middle domain non-covalent ‘Da:EA’ interactions. End-to-end aligned doublestranded fibrils are formed in coordination with another recently described constitutive self-association site (termed ‘D:D’)that is situated at the outer end of each D domain. The D:D site contributes to end-to-end molecular alignment of fibrin molecules within each fibril strand. Concomitant branching and lateral fibril associations to form thick fibers, lead to formation of the mature fibrin clot.

Fibrinogen ‘Kurashiki’ is a unique congenital dysfibrinogenemia that was identified in a homozygous father and his heterozygous son. The abnormality involves a γ chain amino acid mutation (γ G268E) and is characterized by delayed and abnormal fibrin polymerization.

Immunolocalization studies of neonate and older human tissues have previously demonstrated that fibrillin-1 is a component of elastin and non-elastin associated microfibrils. In sections taken from fixed, dehydrated and embedded tissue, microfibrils appear in the transmission electron microscope as hollow rods, 6-8 nm in diameter. When isolated from tissue by homogenization and observed following negative staining or rotary shadowing, microfibrils appear as beaded strings with a degree of extendibility. Recently, a closely related glycoprotein, fibrillin-2, has been described, which is expressed in early fetal development, prior to fibrillin-1, but then disappears in most tissues just prior to birth. We demonstrate here the characterization monoclonal antibodies specific for fibrillin-2. The antibodies are shown by ELISA and immunoblots to be fibrillin-2 specific. The matrix from a variety of tissues, including aorta, tendon, and eye are shown to contain fibrillin-2 by immunofluorescence in a pattern similar to that of fibrillin-1.

Newborn, premature infants have greater susceptibility to infection compared to older newborn infants. Although the reason for greater susceptibility to bacterial infection in premature infants is unknown, there are many reports that focus on polymorphonuclear leukocytes (neutrophils) as the defective cell type in the immature neonatal system. However, we have recently reported that in an rat model of inflammation, neutrophils from adult rats failed to migrate efficiently in neonatal rats. Furthermore, we found that this defect was associated with decreased expression of P-selectin on the surface of neonatal endothelial cells in situ. P-selectin is an adherence molecule that is expressed by activated endothelial cells and serves as the counterligand for CD11/CD18, the β2- integrin that is expressed by activated neutrophils.

We hypothesized that endothelial cell P-selectin expression would be less in human umbilical cord veins from prematurely born (preterm) infants compared to normal term infants. To test this hypothesis, we collected umbilical cords from preterm infants (<27 weeks gestation) and term infants (>38 weeks gestation).

The active form of platelet derived growth factor (PDGF) is a dimeric combination of two polypeptide chains: PDGF-A and PDGF-B. The protein can exist as a heterodimer (PDGF-AB) or as one of two homodimers (PDGF-AA, PDGF-BB), resulting in three isoforms. Two types of PDGF receptors also exist: PDGFRα and PDGFRß. The PDGFRß binds only the PDGF-B chain, while PDGFRα can bind both PDGF-A and -B chains. Both of these receptors show specific temporal and spatial expression patterns that are related to cellular responses including mitogenesis, cell migration, chemotaxis, adhesion and differentiation in a variety of cell types.

Spontaneous mutants in which the PDGFRα is absent exhibit fatal defects during fetal development in mice. Some of these malformities include cardiac septal, valve, and outflow tract defects. Similar defects occur when the function of PDGF-A is disrupted by blocking antibodies. Disruption of PDGF-B function, results in ventricular and atrial dilation, hyper-trabeculation and reduction of the ventricular wall to the thickness of a single cell layer.